Discovery of New Hepatitis B Virus Capsid Assembly Modulators by

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Discovery of New Hepatitis B Virus Capsid Assembly Modulators by an Optimal High-throughput Cell-based Assay Yameng Pei, Chunting Wang, Haijing Ben, Lei WANG, Yao Ma, Qingyan Ma, Ye Xiang, Linqi Zhang, and Gang Liu ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.9b00030 • Publication Date (Web): 14 Feb 2019 Downloaded from http://pubs.acs.org on February 15, 2019

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ACS Infectious Diseases

Discovery of New Hepatitis B Virus Capsid Assembly Modulators by an Optimal High-throughput Cell-based Assay

Yameng Pei1,#, Chunting Wang1,#, Haijing Ben2, Lei Wang3, Yao Ma1, Qingyan Ma1, Ye Xiang3, Linqi Zhang2,*, Gang Liu1,*

1, School of Pharmaceutical Sciences, Tsinghua University, Renhuan Building, Rm 311, Beijing, 100084, China. 2, School of Medicine, Comprehensive AIDS Research Center, and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Tsinghua University，Medical Sciences Building, Suite A209, Beijing 100084, China. 3, Beijing Advanced Innovation Center for Structural Biology, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Center for Global health and Infectious Diseases, Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Medical Sciences Building, Suite A207, Beijing 100084, China. Corresponding author: Gang LIU, Email:[email protected] Keywords ： HBV, High-throughput screening, HepAD38-luc cell line, Capsid assembly modulator, Chemical library

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In this article, a simple and effective high-throughput screening (HTS) assay was developed to identify anti-HBV compounds by using a HepAD38 luciferase reporter (HepAD38-luc) cell line that can effectively exclude the false positive hit compounds targeted on the tetracycline off (tet-off) regulation system. Through screening in-house chemical libraries, N-phenylpiperidine-3-carboxamide derivatives, represented by 1 and 2 were identified, while the other false positive hits, i. e. quinoxaline (3) and benzothiazin (4) derivatives were simultaneously excluded. Compound 1 and 2 exhibit strong inhibitory activity against HBV replication both in HepAD38 and HepG2.2.15 cells. Further studies revealed that 1 and 2 reduced extracellular HBV DNA, HBeAg and intracellular HBV intermediates, including total DNA, RNA and precore RNA of HBV. Size exclusion chromatography (SEC) and electron microscopy (EM) investigations demonstrated that 1 and 2 remarkably induce the formation of morphologically intact capsids and accelerate the dynamics of capsid assembly ， suggesting that both 1 and 2 were type I capsid assembly modulators (CAMs).

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Hepatitis B virus (HBV) infection gravely threatens the world population. Chronic hepatitis B (CHB) patients face a high risk of developing liver fibrosis, cirrhosis and even hepatic carcinoma (HCC). According to the latest survey released in 2018 by the WHO,1 more than 257 million people were living with HBV, and approximately 887,000 people died of liver cirrhosis and HCC caused by CHB in 2015. HBV is a member of the hepadnaviridae family and has a 3.2 kb circular, partially doublestranded DNA genome. The life cycle of HBV involves eight important processes, including entry, uncoating, nuclear import, transcription, nucleocapsid assembly, reverse transcription and viral secretion out of host liver cells. HBV enters into hepatocytes through its receptor, sodium taurocholate co-transporting polypeptide (NTCP).2 After uncoating its nucleocapsid, the relaxed circular DNA (rcDNA) is released, transported into the nucleus and repaired into covalently closed circular DNA (cccDNA).3-4 Taking cccDNA as a template, four distinct viral transcripts [pregenomicRNA (pgRNA), preS1, preS2, and X RNAs] are synthesized and translated into seven viral proteins.5 Core protein (HBcAg) and viral polymerase are from pgRNA, while secretory HBV e antigen (HBeAg) is from precore RNA that is a few bps longer upstream than pgRNA.6 HBX protein, a regulatory protein, is from X RNA. The HBV surface antigen (HBsAg) consists of three envelope proteins, including L protein from preS1 RNA and M and S proteins from preS2 RNA.7-8 After finished above steps, viral polymerase and pgRNA are encapsulated into the nascent nucleocapsid which was formed through self-assembly of HBV core protein.7,

9

Subsequently, the mature

nucleocapsids containing rcDNA either re-enter the cell nucleus for another cycle or 3 / 37



are enveloped to package infectious viral particles that are secreted out of hepatocytes.45, 7 Every above step in the HBV life cycle could be a target for anti HBV drug discovery.

However, until recent, only nucleos(t)ide analogues (NAs) and interferon-α were approved for HBV patient treatment. NAs can reduce HBV copies in plasma but seldom eliminate HBV DNA. A large portion of patients developed resistance to NAs, especially lamivudine, after long-term treatment.10 Interferon-α inhibits the virus by boosting the immune response to HBV. The insufficient efficiency11 and poor tolerance12,13,14 limited its use in clinic. Unfortunately, there were no significantly synergistic effects shown in the combination of interferon-α and NAs.5, 11, 15 In order to develop an effective HTS assay for anti-HBV drug discovery, we selected HepAD38 cell line16 that can generate higher level of HBV copies comparing to HepG2.2.15 cell line17. The drawback of this cell line in anti-HBV screening is that the transcription of HBV genome is driven by a tetracycline off (tet-off) regulation system16, 18,

which may lead false positive hits that inhibit tetracycline-regulated CMV promoter

initiation. In this article, we successfully constructed a HepAD38 luciferase reporter (HepAD38-luc) cell line that simultaneously expresses high-level HBV copies and a luciferase reporter controlled by the tetracycline-regulated CMV promoter.16, 19 Thus, false positive hits targeting on tet-off regulation system could be excluded conveniently. To perform a HTS for HBV drug discovery, we further found that heat denaturation is a simple, accurate and low-cost method for highly efficient DNA extraction. We then screened an in-house chemical library involving approximately ten thousand compounds. Compounds 1 and 2 (N-phenylpiperidine-3-carboxamide derivatives), 3 4 / 37


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(quinoxaline analog) and 4 (benzothiazin analog) were disclosed. Compounds 1 and 2 inhibited HBV replication in both HepAD38 cells and HepG2.2.15 cells, while 3 and 4 inhibited tetracycline-regulated CMV promoter initiation and were false positive compounds. Further investigation revealed that 1 and 2 reduced the extracellular HBV DNA, HBeAg and intracellular HBV intermediates, i.e., total DNA, RNA and precore RNA. Size elution chromatography (SEC) and electron microscopy (EM) studies demonstrated that both 1 and 2 were type I capsid assembly modulators (CAMs) that accelerate HBV capsid assembly and induce the formation of morphologically intact capsids. These results proved that our HTS assay is valid to discover anti-HBV agents, and that two new CAMs are expected to provide new structure scaffolds for the development of anti-HBV drugs. Results Development of a high-throughput screening assay for identification of anti-HBV agents. To develop an HTS assay for anti-HBV drug discovery, we selected HepAD38 cell line that can generate nearly two log10 higher copies of HBV DNA than that generate in HepG2.2.15 cells (Figure 1A and 1B). High level HBV copies could amplify the dynamic detection range. For instance, 100M lamivudine treatment reduced the HBV copies by about one log10 value in HepG2.2.15 cells vs. about 1.5 log10 value in HepAD38 cells (Figure 1B). Due to time consuming, expensive cost and intensive labor work, HBV DNA extraction is another critical process and rate-limiting step that limits the application of HTS assay. According to the comparing experiments, we found that the extraction efficacy of pronase lysis method is lower than that of alkali 5 / 37



lysis combined heat denaturation, pronase lysis combined heat denaturation and heat denaturation alone (Figure 1B). Taking into account convenience, speed and accuracy, we selected heat denaturation method in our HTS assay. Results disclosed that heat denaturation alone for 15 min at 95 ℃ could optimally expose viral DNA up to 5.5107 copies (Figure 1C). HBV gene expression in HepAD38 cells was regulated by a tet-off regulation system (Figures 1E and 1F). On the other word, if chemicals (i.e. tetracycline known as a tetoff regulation system inhibitor) block the interaction between tTA and CMV promoter via binding to tTA, the HBV gene transcription will be suppressed and false positive results will be reported. To exclude the false positive results, we then constructed a cell line, HepAD38-luc, derived from HepAD38 stably transfected with a pLVX-TightPuro-Luciferase reporter regulated by the existing tTA transcription factor. The HepAD38-luc cell line, similar to HepAD38 cells, retained its high HBV DNA levels and sensitivity to lamivudine (Figure 1D). Figure 2 depicted the developed HTS based on HepAD38-luc cell line. In this assay, both HBV DNA copies and luciferase activity were simultaneously detected. Therefore, compounds reducing HBV DNA copies without luciferase activity inhibition were identified as anti-HBV positive hits. One thousand and sixty-one compounds were randomly selected to calculate the Zfactor value, which was an index of the quality of an HTS assay.20 As shown in Figure 4E, the Z value was an average of 0.61, which validates the HTS is suitable for hits identification (1>Z≥0.5 is regarded as an excellent assay)20. Identification of anti-HBV agents. An in-house chemical library containing 6 / 37


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approximately ten thousand compounds was screened by employing this HTS (Figure 2). Two compounds (1 and 2, Figure 3) sharing N-phenylpiperidine-3-carboxamide scaffold (Table 2) were identified, while compounds, represented by 3 and 4, sharing 1, 2, 3, 4(4H)-Quinoxaline and 2H-1, 4-Benzothiazin-3(4H)-one scaffolds (Figure 3) were also disclosed. All these four compounds as well as tetracycline, BAY41-4109, AT130 and lamivudine led to significant reduction of the HBV copies in HepAD38-luc cell supernatant without observed cytotoxicity (Figures 4A and 4D). Data further revealed that only 1, 2, BAY41-4109, AT130 and lamivudine did not inhibit the luciferase activity (Figure 4B). In addition, no anti-HBV activity of tetracycline, 3 and 4 was observed in HepG2.2.15 cells (Figure 4C) and 1 and 2 effectively decreased the level of HBV DNA both in HepAD38 and HepG2.2.15 cell supernatants (Figures 4C and 5A). The above proved 1 and 2 were anti-HBV positive hits and 3 and 4 were false positive compounds, suggesting this assay can accurately distinguish anti-HBV compounds and false positive compounds caused by tet-off regulation system. AntiHBV EC50 of 1 and 2 were 0.02±0.007 µM and 0.35±0.16 µM in HepAD38 cells and 0.05±0.007 µM and 0.48±0.16 µM in HepG2.2.15 cells, respectively (Table 1). Compound 2 had no visible cytotoxicity up to 100 µM and the CC50 value of 1 was about 35 µM both in HepAD38 and HepG2.2.15 cells (Table 1). Several analogues of 1 and 2 with varied substituents at R1 position was then designed and synthesized (513, Table 2). Although anti-HBV potency of these compounds were not improved, structure optimization is continuously ongoing in our lab. Target identification of compound 1 and 2. In order to identify the target of 1 and 2, 7 / 37



biomarkers of HBV life cycle were detected after treatment for 4 days in HepAD38 cells. All the tested compounds decreased the secreted HBV DNA, with 80% suppression achieved at 3 µM (Figure 5A). HBeAg was reduced by approximately 50 % after treatment with 1, 2, BAY41-4109 and AT130 excepted for lamivudine, consistent with the decrease of intracellular precore mRNA that translated to HBeAg (Figure 5B, 5E). None of these compounds influenced HBsAg level (Figure S1A), which suggested that the secretion of the virus may not be affected. Thus, it is reasonable to speculate that 1 and 2 may affect HBV replication process before the virus secretion. At the intracellular level, different from lamivudine that only decreased DNA level, BAY41-4109, AT130, 1 and 2 reduced intracellular DNA, RNA and precore RNA level (Figure 4C, 4D and 4E), indicating their different mechanism from reverse transcriptase inhibitors, i. e. lamivudine. Unfortunately, all these compounds did not eliminated cccDNA in HepAD38 cells through 4 days treatment (Figure S1B). In addition, compound 1 and 2 also did not inhibit HBV core promoter activity compared to positive control helioxanthin 8-121 (Figure S1C). Capsid assembly is a critical cytoplasmic process of HBV replication. CAMs are usually classified into two types. Type I CAMs (represented by phenylpropenamide (PPA) derivative AT13022 and sulfamoylbenzamide (SBA) derivatives23, 24) induce the formation of morphologically intact capsids, and Type II CAMs (e.g., heteroarypyrimidine (HAP) derivative BAY41-410925) misdirect core protein dimers to assemble abnormal structures. We investigated the effect of 1 and 2 on capsid assembly using SEC and EM technologies. Ten µM compounds were individually 8 / 37


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incubated with 5µM recombinant HBV core protein (amino acids (aa) 1 to 149, cp149) overnight before SEC and EM. It appeared two fraction-peaks in DMSO control (Figure 6A), indicating the co-existence of self-assembling capsids (left peak, early fraction) and core protein dimers (right peak, late fraction). We found that BAY41-4109 induced abnormal capsids with earlier retention time of left peak than the one in DMSO, and that AT130 induced morphologically intact capsids with the same retention time to the one in DMSO (Figure 6A and 6C). The capsids peak position in SEC of both compound 1 and 2 were as the same as the type I CAM AT130 (Figure 6B). EM images disclosed the formation of morphologically intact capsids of 1 and 2 (Figure 6C). Therefore, it was concluded that 1 and 2 are type I CAMs. The ability to induce capsid assembly of 1 was stronger than that of 2 because of higher ratio of capsid to dimers in SEC (4.5 versus 0.83, Figure 6B). This result was also consistent with their different EC50 values (1: 0.02±0.007µM and 2: 0.35±0.16µM) against HBV. Discussion Although HepG2.2.15 cells17 was generally used for anti-HBV compounds screening in low throughput, the narrow dynamic range of HBV DNA level by real time qPCR detection often results into ambiguous data in our laboratory even using HepG2.2.15 cell line from different laboratories (data not show). It is attractive that the HBV copies of HepAD38 cell line are 100-fold higher than that of HepG2.2.15 cells under the same amplification condition. The high HBV copies amplify the dynamic range to approximately 1.5 log HBV copies after compounds treatment, ensuring the authenticity and reproducibility of the experiment. In addition, a simple heat 9 / 37



denaturation extraction method of HBV DNA was first successfully applied in HTS assay for anti-HBV agent discovery, increasing the performance of this HTS assay. In the primary anti-HBV screening, we carried out HTS based on HepAD38 cells. However, the extremely high positive rate (~10 percent) from quinoxaline and benzothiazin derivatives alerted us something wrong with this HTS. We further confirmed that these hit compounds were not active against HBV in HepG2.2.15 cells. Considering mean value (0.61) of Z-factor of randomly selected 1061 compounds, the reproducibility and quality of HTS was excellent (1>Z≥0.5).20 It is noted that HBV expression in HepAD38 cells was regulated by a tet-off regulation system and chemicals interfering tetracycline-responsive CMV promoter in HepAD38 cells may inhibit HBV gene transcription and cause false positive results. A counter screen using another cells e.g. HepAD43 cells (expresses β-galactosidase gene under the transcriptional control of tetracycline-responsive CMV promoter) to exclude false positives results18 was needed in HepAD38 cell-based anti-HBV screening. In order to simultaneously report false positive and true anti-HBV activity in one cell line, we optimized our HTS assay by constructing a HepAD38-luc cell line through lentivirus infecting HepAD38 cells. HepAD38-luc cell line expressed luciferase reporter gene while maintaining high HBV copies and anti-HBV sensitivity to lamivudine. These two genes were regulated by tet-off regulation system synchronously and compounds reducing HBV copies through interfering tetracycline-responsive CMV promoter can be reported by the reduction of luciferase activity. We successfully performed two readouts in this HTS containing anti-HBV activity through detecting HBV copies of 10 / 37


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HepAD38-luc cells supernatant and exclusion of false positive results through detecting luciferase activity of residual cell lysis after compounds incubation for six days. Compounds reducing HBV DNA without luciferase activity inhibition were potential anti-HBV hits. Quinoxaline and benzothiazin derivatives that targeted on tet-off regulation system were identified from our small molecular chemical libraries, however, Nhenylpiperidine-3-carboxamide compounds were disclosed as anti-HBV chemical scaffold. Both HBeAg and HBsAg are important biomarkers of HBV infection.7 Completely elimination of HBsAg is considered as functional cure of HBV infection in clinic.5, 2628

HBeAg reduction is a symbol of sustained therapeutic response.29 In this study, it

was found that HBeAg level of HepAD38 cells was significantly reduced after BAY414109, AT130, 1 and 2 treatment for 4 days but not lamivudine. This result was consistent with the reduction of precore RNA, which can be translated to HBeAg after compounds treatment. The secretion of HBsAg was not suppressed by BAY41-4109, AT130, 1 or 2 treatment in HepAD38 cells. All above concluded 1 and 2 targeted on intracellular HBV replication cycle but not viral secretion. Through detection of intracellular HBV replication intermediates, we found that BAY41-4109, AT130, 1 and 2 were able to significantly reduce HBV DNA, RNA but not cccDNA in tested HepAD38 cells. CAMs could reduce the level of core-associated viral RNA by interfering with the packaging of pgRNA into the immature core particle18 that might result into a decreased total cytoplasmic RNA (including encapsidated RNA). We 11 / 37



observed a moderate reduction of total mRNA in HepAD38 cells, which was consistent with that the AT-61, an AT-130 analogue, reduced the cytoplasmic viral RNA in HepAD38 cells after treatment for 3 days18. Alternatively, Berke et al. 34, 35 also reported that CAMs, i.e. BAY41-4109 or AT130, decreased the total mRNA in a HBV in vitro infection system using HepaRG and PHH cells. Nevertheless, the more authoritative HBV infection system will be used in our further research to discuss the observation of these identified new CAMs. We also tested whether compounds affected HBV promoter initiation and the result indicated that 1 and 2 were inactive. Next, we investigated the interactions between 1, 2 and HBV core protein. SEC and EM studies revealed that 1 and 2 effectively induced the formation of morphologically intact HBV capsids and accelerated capsid assembly. Compared to AT130 (Type I CAM) and BAY41-4109 (Tpye II CAM), capsids peak positon of 1 and 2 in SEC were as the same as AT130, which indicated that 1 and 2 were type I CAMs. SEC ratio of compound 1 (4.5) was higher than 2 (0.83) which was consistent with the stronger anti-HBV activity of 1 than 2. Conclusion We developed an anti-HBV drug discovery HTS assay based on HepAD38-luc cell line that was able to report anti-HBV activity of chemicals and simultaneously exclude false positive results caused by interfering tet-off regulation system. Compound 1 and 2 were disclosed as anti-HBV lead compounds by this study. Primary investigation revealed that 1 and 2 accelerated the formation of HBV normal capsids and reduced intracellular HBV RNA and DNA. Discovery of 1 and 2 give a new research direction of chemical 12 / 37


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structure for next generation of anti-HBV CAMs. Experimental Section Cell Lines and Culture Conditions HepG2.2.15, HepAD38, HepAD38-luc, HepG2 and 293T cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % (v/v) fetal bovine serum (FBS) and penicillin-streptomycin solution (100×) at 37 ℃ with 5 % CO2. HepG2.2.15 and HepAD38 cells were routinely checked for resistance to G418 (Invivogen, ant-gn-1) at 500 μg/ mL. HepAD38 cells (1×106 cells/well in a 6-well plate) were infected with 500 µL pLVX-tight-puro luciferase lentivirus (Clontech, 632162) and selected in DMEM containing 10 % FBS, 2 μg/mL puromycin and 500 μg/mL G418 at approximately 24 h post-transfection. An infinite dilution was performed to obtain cell clones after control group cells (HepAD38 cells without infection) were all dead at 2 μg/mL puromycin, and monoclonal cells were called HepAD38-luc. Compounds and Plasmids Pzac1.2HBV plasmid30 (Gene bank, AY123424), psPAX2 (Addgene, 12260), pMD2.G (Addgene, 12259) and pLVX-tight-puro-luciferase reporter plasmid (Clontech, 632162) were stored in our laboratory. Tetracycline and lamivudine were purchased from Meilunbio. Helioxanthin 8-1 was purchased from MedChemExpress. Anti- HBV Screening Assay Design Culture media containing 5 % FBS in DMEM was used for the anti-HBV assay. HepAD38-luc cells were seeded into 96-well culture plates at approximately 1×104 13 / 37



cells/well in a volume of 100 µL and treated with compounds or DMSO for six days after cells adhered to the well. Medium containing compounds or DMSO were replaced with new after 3 days. A total of 60 μL of cell supernatant was transferred to an eight strip PCR tube using a multichannel pipettor and analyzed for extracellular HBV DNA, HBeAg or HBsAg. HBV Particle DNA Extraction Alkali method31: 60 μL cell culture supernatant was mixed equally with 7 μL alkali medium (1 mol/L NaOH, 20 mol/L NaCl, 5 % SiO2) and then incubated for 15 min at 95 °C in a PCR amplifier followed by centrifugation for 10 min at 4000 rpm. Pronase method: 3 μL pronase (Roche, 10165921001) was added to 60 μL cell culture supernatants accordingly, and the mixture was incubated at 37 °C for 30 min. Heating: 60 μL samples were incubated for 15 min at 95 °C in a PCR amplifier and then centrifuged for 10 min at 4000 rpm. All of these samples were stored at 4 ℃. Compound Cytotoxicity Test Compound toxicity was detected by Cell Counting Kit-8 (CCK8, Dojindo, CK04-05） as directed by the manufacturer. Briefly, approximately 1×104 cells were seeded in 96well culture plates and treated with compounds or DMSO for six days after cell adherence. Among the six day treatment period, the culture medium with test compounds was replaced with new media containing compounds at 72 h intervals. At day six of compound treatment, cells were incubated with 50 μL of compound screening medium containing 20 % CCK-8 for approximately 30 min at 37 ℃ and 5 % CO2. OD values were read at 450 nm. For cytotoxicity CC50, the data for each well was calculated 14 / 37


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using the following formula: 100 % ×(OD control-

value of control-

OD

value of samples)/

(OD

value of

OD value of blank), and analyzed using a 3- or 4-parameter curve fitting algorithm in

Graph Pad Prism. Intracellular DNA and RNA Extraction Cells were plated in 12-well culture plates with a density of 2 × 105 cells/well and cultured with fresh medium containing compounds (3 μM) or DMSO for four days after cell adherence. The media was replaced every two days, and the cells were collected and stored at -80 ℃. Both intracellular DNA and RNA were extracted using the TIANamp Genomic DNA Kit (Tiangen, DP304) and TRIzol according to the manufacturer’s instructions. RNA was reverse transcribed into cDNA using the HighCapacity cDNA Reverse Transcription Kit (ThermoFisher, 4368813) as directed by the manufacturer. For cccDNA extraction, 5 L eluted DNA(100-400ng/uL), 1 L 10× reaction buffer, 1 L PSD (10U, Epicentre, 3101K) and 3 L water at 37℃ for 1 hour and 70℃ for 20 minutes were subjected for cccDNA detection. Quantitative PCR and Quantitative Reverse Transcription -PCR HBV Particle DNA Detection Twenty microliter reaction systems contained 10 μL TaqMan® Gene Expression Master Mix (Applied Biosystems, 4369016), 2μL forward and reverse primers (10 μM), 0.25 μL probe, 5.75 μL dd H2O and 2 μL treated samples. PCR conditions: 95 °C for 10 min, 95 °C for 15 s, and 60 °C for 1 min for 40 cycles in a real-time 7500 machine. HBV Forward primer: 5’-CCAAATGCCCCTATCCTATCA-3’; and HBV Reverse primer:

5’-GAGGCGAGGGAGTTCTTCTTCTA-3’.

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HBV

probe:

5’-


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CGGAAACTACTGTT GTTAGACGACGAGGCAG-3’. Pzac-1.2HBV plasmid30 was used as the standard substance to draw the standard curve, and 80 ng/mL plasmid corresponded to a 1013 HBV DNA load. The standard curve contained eight points from 1011 to 104 with a 10fold dilution. For anti-HBV EC50, the data for each well was calculated using the following formula: 100 % × (1-2-(CT

samples-C control)), T

and analyzed using a 3- or 4-

parameter curve fitting algorithm in Graph Pad Prism. Intracellular DNA and RNA Detection The comparative CT (ΔΔCT) quantitation method was used in quantitative PCR (qPCR) or quantitative reverse transcription (qRT-PCR) detection. For intracellular DNA and cccDNA detection, PCR reaction systems were the same as above. In addition, GAPDH was used as a housekeeping gene and 20 μL reaction systems include 10 μL TaqMan® Gene Expression Master Mix (Applied Biosystems, 4369016), 1 μL primer mix (ThermoFisher, Hs02758991_g1), 2 μL DNA samples and 7 μL dd H2O. cccDNA former

primers:

5’-CCCCGTCTGTGCCTTCTC-3’;

reverse

primers:

5’-

CAGCTTGGAGGCTTGAACAGT-3’; cccDNA probes: 5’-FAM-ACTCTCAGCAAT GTCAACGACCGACC-TAM-3’. For intracellular RNA detection, 20 μL reaction systems consist of 10 μL Power SYBR® Green PCR Master Mix (Applied Biosystems, 4367659), 2 μL forward and reverse primers (10 μM), 2 μL DNA samples and 6 μL ddH2O. GAPDH forward primer: 5’-ACCCACTCCTCCACCTTTG-3’; and reverse primer: 5’-CTGTAGCCAAATTCGTTGTCAT-3’. pgRNA forward primer: 5’TTTTCACCTCTGCCTAATCATCTCTTG-3’; precore RNA forward primer: 5’16 / 37


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TTGGTCTGCGCACCAGCACC-3’; and their common reverse primer: 5’-GAA GGAAAGAAGTCAGAAGGCAA-3’.6 The data for each well was calculated using the following formula: 2-((CT sample-CT sample GAPDH) – (CT control-CT control GAPDH)), and analyzed in Graph Pad Prism. Luciferase Activity Detection 1×104 AD38-luc cells were seeded in 96 well plate and cultured with compounds (3 µM) for 6 days. Then, cells were collected and detected luciferase activity by Fire-Lucy Assay Kit (Vigorousbio, T003) as directed by manufacturer’s instructions. Relative luciferase activity (S/VC) indicates the sample value/vehicle control value. Enzyme-Linked Immunosorbent Assay HepAD38 cell culture supernatant was diluted 3-fold for HBeAg detection (Kehua, 20123400740) and 5-fold for HBsAg detection (Kehua, S10910113) according to manufacturer’s instructions. Enzyme-Linked immunosorbent assay (ELISA) results are presented as S/VC (S = sample OD value and VC = vehicle control OD value) Z-factor Calculation A total of 1061 compounds were randomly selected, which were distributed in 63 screening results, and every time result included approximately 17 compound screening data in a 96-well plate. Z values were calculated by the equation 20: Z = 1-(3×SD of sample+3×SD of control)/(mean of sample –mean of negative control). SEC and EM studies. A recombinant HBV core protein (Cp149) was used in SEC and EM studies. Determination of compounds effects on capsid formation were performed as previously 17 / 37



described

32-33with

some modifications. Briefly, 5μM Cp149 proteins was incubated

with 10μM compounds in a buﬀer containing 150 mM NaCl, 50 mM HEPES (pH7.4) at 37 °C overnight prior to negative staining and SEC. For SEC, samples were analyzed by an Äkta purifier with a size exclusion column (GE Healthcare, Superdex 200 Increase 5/150 GL (micro)) and running buffer contained 100 mM Tris, 100 mM NaCl, 17g/L Sucrose adjusted to pH 7.5. The UV absorbance curve was measured at 280 nm and the ratio of area under the curve (AUC) was calculated to assess capsid formation induced by compounds. For EM, Samples were adsorbed to freshly glow discharged carbon coated grids for 30s. After drying by filter paper, stained in fresh 1% (wt/vol) uranyl acetate acid for 1 min. After absorbing excess water by filter paper and drying, the grids were examined with transmission electron microscope. Statistics Statistical significance was shown by p-values, which were calculated by t-test, and unpaired samples for the mean. Supporting Information Evaluation of extracellular HBsAg level, intracellular HBV cccDNA and HBV core promoter activity; experimental procedures for HBV core promoter activity detection and information of the compounds. Author Information Author Contributions # Y.P.

and C.W. contributed equally to this work.

Acknowledgment 18 / 37


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We thank the National Natural Science Foundation of China (No. 81773575, 81573289, 81161120402) for support. Abbreviations aa, amino acids; AUC, area under the curve; CAMs, capsid assembly modulators; cccDNA, covalently closed circular DNA; CHB, chronic hepatitis B;

ELISA,

enzyme-linked immunosorbent assay; EM, electron microscopy; HBV, hepatitis B virus; HBeAg, HBV e antigen; HBsAg, HBV surface antigen; HCC, hepatic carcinoma; HTS, high-throughput screening; min, minutes; NAs, Nucleos(t)ide analogues; nt, nucleotides;

NTCP,

sodium

taurocholate

cotransporting

polypeptide;

PCR,

polymerase chain reaction; pgRNA, pregenomic RNA; Pol-pgRNA, polymerasepregenomic RNA; PPA, phenylpropenamide; qPCR, quantitative PCR; qRT-PCR, quantitative reverse transcription PCR; rcDNA, released relaxed circular DNA; SBA, sulfamoylbenzamides; SEC, size exclusion chromatography; tet-off, tetracycline off; VC, vehicle control. Reference (1) Hepatitis B http://www.who.int/news-room/fact-sheets/detail/hepatitis-b. (2) Yan, H., Zhong, G. C., Xu, G. W., He, W. H., Jing, Z. Y., Gao, Z. C., Huang, Y., Qi, Y. H., Peng, B., Wang, H. M., Fu, L. R, Song, M., Chen, P., Gao, W. Q., Ren, B. J., Sun, Y. Y., Cai, T., Feng, X. F, Sui, J. H., Li, W. H., (2012) Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. Elife 1. DOI: 10.7554/eLife.00049 (3) Hu, J. M., Liu, K. C., (2017) Complete and Incomplete Hepatitis B Virus Particles: 19 / 37



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targets. Hepatol Int. 5, 644-53. DOI: 10.1007/s12072-011-9261-3. (10) Suzuki, F., Hosaka, T., Suzuki, Y., Akuta, N., Sezaki, H., Hara, T., Kawamura, Y., Kobayashi, M., Saitoh, S., Arase, Y., Ikeda, K., Kobayashi, M., Watahiki, S., Mineta, R., Kumada, H., (2014) Long-term efficacy and emergence of multidrug resistance in patients with lamivudine-refractory chronic hepatitis B treated by combination therapy with adefovir plus lamivudine. J. Gastroenterol. 49, 1094-1104. (11)Wursthorn, K., Lutgehetmann, M., Dandri, M., Volz, T., Buggisch, P., Zollner, B., 20 / 37


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Figure legends Figure 1. HBV copies detection, different DNA extraction methods evaluation and diagram of HBV genome and tet-off regulation system in HepAD38 cells. A, Comparison of HBV copies in HepAD38 and HepG2.2.15 cell supernatant. B, Pronase lysis, pronase lysis combined heat denaturation, alkali lysis combined heat denaturation and heat denaturation alone methods used for DNA extraction were assayed in HepAD38 cells after treatment with Lamivudine; HBV DNA of HepG2.2.15 cell supernatant was extracted by heat denaturation method after dose-dependent Lamivudine treatment. C, Heat denaturation time of 15 min at 95℃ was enough for HBV DNA extraction in HepAD38 cell supernatant. D, HBV copies and anti–HBV detection sensitivity of HepAD38 cells and HepAD38-luc cells after treatment with dose-dependency Lamivudine. Cells (1.0 × 104) were seeded in 96-well plates and cultured for six days with DMSO or lamivudine at 100, 10, 1, 0.1, 0.01, and 0.001 μM for six days. Cell supernatant was detected by qPCR and data points represent the mean of 3 culture wells ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. E, There is a tet-off regulated CMV promoter at upstream of HBV genome containing four open reading 25 / 37



frames in HepAD38 cells. F, Tet-off regulation system causes false positive result through interfering with the binding of tTA and CMV promoter.

Figure 2. A diagram of anti-HBV HTS assay based on HepAD38-luc cell line. Briefly, 1104 cells were seeded in 96-well plates and incubated with compounds for 6 days. The cell supernatant was then heated in parallel in a PCR amplifier for 15 min at 95 ℃ and centrifuged for 10 min at 4000 rpm. Two microliters treated supernatant as a PCR template was amplified for DNA detection. For the active compounds, cells remaining in the 96-well plates was lysed and luciferase activity was detected using a Fire-Lucy Assay Kit. Therefore, compounds that have anti-HBV activity and no effect on luciferase activity were anti-HBV hit.

Figure 3. Chemical structures of newly discovered compounds.

Figure 4. Optimized anti-HBV HTS assay can accurately and excellently distinguish anti-HBV compounds from false positive compounds. A, HBV copies was decreased after treated with Compound 1, 2, 3, 4, tetracycline, BAY41-4109, AT130 and lamivudine in HepAD38-luc cells. B, Compound 3, 4 as well as tetracycline inhibited luciferase activity at the treated concentration. C, Compound 1, 2 as well as BAY414109, AT130 and lamivudine reduced HBV DNA in HepG2.2.15 cells supernatant, but 3, 4 and tetracycline was not. D, All compounds were non-cytotoxic to HepAD38-luc cells at tested concentrations using a CCK-8 kit. A-D: Cells were seeded with 26 / 37


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HepAD38-luc cells of 1×104 cells/well in 96-well plate and HepG2.2.15 cells of 2× 105 cells/well in 12-well plate and cultured with compound (3 µM) or DMSO for six days. The supernatant was then collected for qPCR detection of HBV DNA. The remaining HepAD38-luc cells in 96-well plate were cultured with 50µL media including 20 % CCK-8 for cytotoxicity detection. Data points represent the mean of 3 culture wells ± SD. ***p < 0.001, ****p < 0.0001 VC represents vehicle control. E. The Z-factor value of total 1061 compounds is an average of 0.61. Every point (batch) represents a Z value mean of 17 screened compounds in a 96-well plate.

Figure 5. Evaluation of extracellular and intracellular HBV replication biomarkers after treated with compounds in HepAD38 cells. Extracellular HBV DNA (A) and intracellular HBV DNA (C) were strongly reduced after compounds treatment. All treated compounds reduced significantly HBeAg (B), total RNA (D) and precore RNA (E) except lamivudine. A-E, A total of 2×105 cells were plated in 12 well plates and cultured with compounds (3 µM) or DMSO for four days. Cell supernatant was collected for qPCR or ELISA, and the remaining cells were collected to extract RNA or DNA. All these DNA or cDNA samples were detected by qPCR or qRT-PCR. Data points represent the mean of 3 culture wells ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. VC represents vehicle control.

Figure 6. Capsid assembly reaction examined by SEC (A), SEC ratio (B), EM（C） study using the protocols as described in Methods. SEC ratio means Capsids AUC/Core 27 / 37



Page 28 of 38

Protein Dimers AUC.

Table 1. Anti-HBV activity, cytotoxicity and selectivity index (SI) of compounds HepAD38 Cell Line

HepG2.2.15 Cell Line

Compounds EC50(µM)

CC50(µM)

3TC

0.02±0.004

>100

AT130

0.2±0.07

BAY41-4109

0.06±0.01

SI

EC50(µM)

CC50(µM)

SI

5000 0.01±0.002

>100

10000

>100

500

0.14±0.03

>100

714

20.78±7.90

346

0.02±0.006

8.96±1.18

448

1

0.02±0.007 35.05±8.63 1752 0.05±0.007 35.06±14.79

701

2

0.35±0.16

208

>100

285

0.48±0.23

>100

*EC50: Compound concentration that suppresses 50 % virus growth. CC50：Compound concentration that suppresses 50 % cell growth. SI50: CC50/ EC50.

28 / 37


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Table 2. Structure activity relationship of N-phenylpiperidine-3-carboxamide derivatives. F Cl

HN

O

N

R

Compound

R

HepAD38 EC50 (µM)

HepAD38 CC50 (µM)

0.02±0.007

35.05±8.63

0.35±0.16

>100

1.54±0.22

≈100

2.04

12.80

0.26

37.5

0.86

100

0.86

>100

H N

1 O

O

2 O N

O

O

5

H N

6

O

O

7 O O

8 N O

O

9

N

29 / 37



Page 30 of 38

O

10

0.97

13.74

>10

ND

>10

ND

2.70

>100

Cl O

11 O

12 O

O

13 O

30 / 37


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Figure 1

31 / 37



Figure 2

32 / 37


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Figure 3 F

F Cl

Cl N

HN

O

HN

O

NH N

N O N

O 1

O 2

Cl F

HN S O O O

N H

S

N

N O

O

HN S O O O

Cl 3

Figure 4

33 / 37


4

N H

O


Figure 5

34 / 37


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Figure 6

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Table of contents graphic (TOC) 36 / 37


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Table of contents graphic (TOC)


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Discovery of New Hepatitis B Virus Capsid Assembly Modulators by

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